Barley Yellow Dwarf Virus RNA Research Articles

Eukaryotic translation initiation factor 4G (eIF4G) coordinates interactions with eIF4A, eIF4B, and eIF4E in binding and translation of the barley yellow dwarf virus 3′ cap-independent translation element (BTE)

Barley yellow dwarf virus RNA, lacking a 5' cap and a 3' poly(A) tail, contains a cap-independent translation element (BTE) in the 3'-untranslated region that interacts with host translation initiation factor eIF4G. To determine how eIF4G recruits the mRNA, three eIF4G deletion mutants were constructed: (i) eIF4G601-1196, containing amino acids 601-1196, including the putative BTE-binding region, and binding domains for eIF4E, eIF4A, and eIF4B; (ii) eIF4G601-1488, which contains an additional C-terminal eIF4A-binding domain; and (iii) eIF4G742-1196, which lacks the eIF4E-binding site. eIF4G601-1196 binds BTE tightly and supports efficient translation. The helicase complex, consisting of eIF4A, eIF4B, and ATP, stimulated BTE binding with eIF4G601-1196 but not eIF4G601-1488, suggesting that the eIF4A binding domains may serve a regulatory role, with the C-terminal binding site having negative effects. eIF4E binding to eIF4G601-1196 induced a conformational change, significantly increasing the binding affinity to BTE. A comparison of the binding of eIF4G deletion mutants with BTEs containing mutations showed a general correlation between binding affinity and ability to facilitate translation. In summary, these results reveal a new role for the helicase complex in 3' cap-independent translation element-mediated translation and show that the functional core domain of eIF4G plus an adjacent probable RNA-binding domain mediate translation initiation.

Cis- and trans-regulation of luteovirus gene expression by the 3′ end of the viral genome

Translation of the 5.7kb luteovirus genome is controlled by the 3′ untranslated region (UTR). Base pairing between regions of the 3′ UTR and sequences kilobases upstream is required for cap-independent translation and ribosomal frameshifting needed to synthesize the viral replicase. Luteoviruses produce subgenomic RNAs, which can serve as mRNA, but one sgRNA also regulates translation initiation in trans. As on all viruses, the 3′ and 5′ ends contain structures that are presumed to facilitate RNA synthesis. This review describes the structures and interactions of barley yellow dwarf virus RNA that facilitate the complex interplay between the above events and result in a successful virus infection. We also present surprising results on the apparent lack of need for some subgenomic RNAs for the virus to infect cells or whole plants. In summary, the UTRs of luteoviruses are highly complex entities that control and fine-tune many key events of the virus replication cycle.

The 3' cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation.

The 3' cap-independent translation element (BTE) of Barley yellow dwarf virus RNA confers efficient translation initiation at the 5' end via long-distance base pairing with the 5'-untranslated region (UTR). Here we provide evidence that the BTE functions by recruiting translation initiation factor eIF4F. We show that the BTE interacts specifically with the cap-binding initiation factor complexes eIF4F and eIFiso4F in a wheat germ extract (wge). In wge depleted of cap-interacting factors, addition of eIF4F (and to a lesser extent, eIFiso4F) allowed efficient translation of an uncapped reporter construct (BLucB) containing the BTE in its 3' UTR. Translation of BLucB required much lower levels of eIF4F or eIFiso4F than did a capped, nonviral mRNA. Both full-length eIF4G and the carboxy-terminal half of eIF4G lacking the eIF4E binding site stimulated translation to 70% of the level obtained with eIF4F, indicating a minor role for the cap-binding protein, eIF4E. In wge inhibited by either BTE in trans or cap analog, eIF4G alone restored translation nearly as much as eIF4F, while addition of eIF4E alone had no effect. The BTE bound eIF4G (Kd = 177 nm) and eIF4F (Kd = 37 nm) with high affinity, but very weakly to eIF4E. These interactions correlate with the ability of the factors to facilitate BTE-mediated translation. These results and previous observations are consistent with a model in which eIF4F is delivered to the 5' UTR by the BTE, and they show that eIF4G, but not eIF4E, plays a major role in this novel mechanism of cap-independent translation.

Subgenomic RNA as a riboregulator: negative regulation of RNA replication by Barley yellow dwarf virus subgenomic RNA 2

Barley yellow dwarf virus (BYDV) generates three 3′-coterminal subgenomic RNAs (sgRNAs) in infected cells. Translation of BYDV genomic RNA (gRNA) and sgRNA1 is mediated by the BYDV cap-independent translation element (BTE) in the 3′ untranslated region. sgRNAs 2 and 3 are unlikely to be mRNAs. We proposed that accumulation of sgRNA2, which contains the BTE in its 5′ UTR, regulates BYDV replication by trans-inhibiting translation of the viral polymerase from genomic RNA (gRNA). Here, we tested this hypothesis and found that: (i) co-inoculation of the BTE or sgRNA2 with BYDV RNA inhibits BYDV RNA accumulation in protoplasts; (ii) Brome mosaic virus (BMV), engineered to contain the BTE, trans-inhibits BYDV replication; and (iii) sgRNA2 generated during BYDV infection trans-inhibits both GFP expression from BMV RNA and translation of a non-viral reporter mRNA. We conclude that sgRNA2, via its BTE, functions as a riboregulator to inhibit translation of gRNA. This may make gRNA available as a replicase template and for encapsidation. Thus, BYDV sgRNA2 joins a growing list of trans-acting regulatory RNAs.

Conservation of RNA structures enables TNV and BYDV 5' and 3' elements to cooperate synergistically in cap-independent translation.

The subgenomic RNA 2 of tobacco necrosis virus A (TNV sgRNA2) encodes the viral coat protein, is unpolyadenylated and presumably uncapped. Here, we show that TNV sgRNA2 is translated cap independently. This cap-independent translation requires the leader and a 140 nt element of the trailer both in wheat germ extract and in tobacco protoplasts. Similar to barley yellow dwarf virus (BYDV), the TNV 5' and 3' elements stimulate translation synergistically. Computer-aided phylogenetic analysis of the secondary structure of the TNV trailer revealed that the 3' translation element is part of a major conserved stem-loop that contains similarities to structures in the BYDV 3' translation element. These data suggest that the translation mechanisms of TNV sgRNA2 and BYDV RNA are related. To further characterize this relationship, we tested whether cooperativity exists between TNV sgRNA2 and BYDV 5' and 3' elements. We found that the TNV sgRNA2 5' element stimulates translation synergistically with the BYDV 3' element in vitro. This finding is the first evidence for conservation of structures that enable a 5'-3' interaction stimulating cap-independent translation.

A -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA.

Programmed -1 ribosomal frameshifting is necessary for translation of the polymerase genes of many viruses. In addition to the consensus elements in the mRNA around the frameshift site, we found previously that frameshifting on Barley yellow dwarf virus RNA requires viral sequence located four kilobases downstream. By using dual luciferase reporter constructs, we now show that a predicted loop in the far downstream frameshift element must base pair to a bulge in a bulged stem loop adjacent to the frameshift site. Introduction of either two or six base mismatches in either the bulge or the far downstream loop abolished frameshifting, whereas mutations in both sites that restored base pairing reestablished frameshifting. Likewise, disruption of this base pairing abolished viral RNA replication in plant cells, and restoration of base pairing completely reestablished virus replication. We propose a model in which Barley yellow dwarf virus uses this and another long-distance base-pairing event required for cap-independent translation to allow the replicase copying from the 3' end to shut off translation of upstream ORFs and free the RNA of ribosomes to allow unimpeded replication. This would be a means of solving the "problem," common to positive strand RNA viruses, of competition between ribosomes and replicase for the same RNA template.

The 3prime prime or minute-terminal structure required for replication of Barley yellow dwarf virus RNA contains an embedded 3prime prime or minute end.

We determined the 3prime prime or minute-terminal primary and secondary structures required for replication of Barley yellow dwarf virus (BYDV) RNA in oat protoplasts. Computer predictions, nuclease probing, phylogenetic comparisons, and replication assays of specific mutants and chimeras revealed that the 3prime prime or minute-terminal 109 nucleotides (nt) form a structure with three to four stem-loops followed by a coaxially stacked helix incorporating the last four nt [(A/U)CCC]. Sequences upstream of the 109-nt region also contributed to RNA accumulation. The base-pairing but not the sequences or bulges in the stems were essential for replication, but any changes to the 3prime prime or minute-terminal helix destroyed replication. The two 3prime prime or minute-proximal tetraloops tolerated all changes, but the two 3prime prime or minute-distal tetraloops gave most efficient replication if they fit the GNRA consensus. A mutant lacking the 3prime prime or minute-proximal stem-loop produced elevated levels of less-than-full-length minus strands, and no (+) strand. We propose that a "pocket" structure is the origin of (minus sign)-strand synthesis, which is negatively regulated by the inaccessible conformation of the 3prime prime or minute terminus, thus favoring a high (+)/(minus sign) ratio. This 3prime prime or minute structure and the polymerase homologies suggest that genus Luteovirus is more closely related to the Tombusviridae family than to other Luteoviridae genera.

A sequence required for −1 ribosomal frameshifting located four kilobases downstream of the frameshift site

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (−1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical −1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known −1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the −1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5′ cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.

Structure and function of a cap-independent translation element that functions in either the 3' or the 5' untranslated region.

Barley yellow dwarf virus RNA lacks both a 5' cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3' UTR, that confers efficient translation initiation at the AUG closest to the 5' end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3'-5' communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3' and 5' UTR contexts. A seven-base loop sequence was needed only in the 3' UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3' UTR.

A positive-strand RNA virus with three very different subgenomic RNA promoters.

Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes. A major step in control of viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. We used barley yellow dwarf virus (BYDV) as a model system to study transcriptional control in a virus with multiple sgRNAs. BYDV generates three sgRNAs during infection. The sgRNA1 promoter has been mapped previously to a 98-nucleotide (nt) region which forms two stem-loop structures. It was determined that sgRNA1 is not required for BYDV RNA replication in oat protoplasts. In this study, we show that neither sgRNA2 nor sgRNA3 is required for BYDV RNA replication. The promoters for sgRNA2 and sgRNA3 synthesis were mapped by using deletion mutagenesis. The minimal sgRNA2 promoter is approximately 143 nt long (nt 4810 to 4952) and is located immediately downstream of the putative sgRNA2 start site (nt 4809). The minimal sgRNA3 core promoter is 44 nt long (nt 5345 to 5388), with most of the sequence located downstream of sgRNA3 start site (nt 5348). For both promoters, additional sequences upstream of the start site enhanced sgRNA promoter activity. These promoters contrast to the sgRNA1 promoter, in which almost all of the promoter is located upstream of the transcription initiation site. Comparison of RNA sequences and computer-predicted secondary structures revealed little or no homology between the three sgRNA promoter elements. Thus, a small RNA virus with multiple sgRNAs can have very different subgenomic promoters, which implies a complex system for promoter recognition and regulation of subgenomic RNA synthesis.

Translational frameshifting by barley yellow dwarf virus RNA (PAV serotype) in Escherichia coli and in eukaryotic cell-free extracts.

The open reading frame (39K ORF) at the 5' end of the genome of barley yellow dwarf virus, PAV serotype (BYDV-PAV), overlaps with a 60K ORF by 13 nucleotides. Several approaches were used to show that the 60K ORF (putative polymerase gene) is translated by a low-frequency frameshift event in which some ribosomes shift into the 60K ORF rather than terminate at the 39K ORF stop codon. A sequence encompassing this region of overlap induced minus one (-1) translational frameshifting in heterologous and native contexts. In Escherichia coli, with the alpha subunit of lacZ used as a reporter gene, the rate of frameshifting caused by the BYDV-PAV sequence was approximately 3%. Amino acid sequencing of the transframe protein confirmed that ribosomes slip into the -1 frame in the overlapping region which includes a consensus shifty heptanucleotide: GGGUUUU. In a wheat germ translation system, BYDV-PAV genomic RNA from virions frameshifted about twice as efficiently as full-length transcripts from a cDNA clone. Frameshifting in rabbit reticulocyte lysates was much lower for either template. The identity of the 99-kDa wheat germ translation product was verified as the transframe protein by immunoprecipitation with antibody specific for the 60K ORF. These results support our previous observations of frameshifting in protoplasts and illustrate a subtle molecular control mechanism between this pathogen and its host cells.

Barley Yellow Dwarf Virus RNA Has a 5'-Terminal Genome-linked Protein

SUMMARY The 5′ terminus of barley yellow dwarf virus RNA did not become labelled with 32P after dephosphorylation was attempted with calf intestinal alkaline phosphatase and subsequent treatment with [γ-32P]ATP and T4 polynucleotide kinase. Treatment of BYDV RNA with 125I-Bolton-Hunter reagent yielded 125I-labelled BYDV RNA, as shown by acid precipitation analysis. Treatment with RNase yielded a protein of approximate M r 17000 that was destroyed by treatment with Pronase and was serologically distinct from BYDV coat protein. BYDV therefore appears to have a genome-linked protein.

Nonradioactive, photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus

Photobiotin was used to prepare biotinylated, nonradioactive nucleic acid probes for the detection of the RNA of barley yellow dwarf virus (BYDV) in plant extracts. A 1.7-kb cDNA of the PAV isolate of BYDV in the plasmid pUC8 vector was biotinylated and then used intact or as sonicated double-stranded DNA fragments. Simple methods were developed for the preparation of partially purified nucleic acid extracts of cereals and their spotting, after formaldehyde treatment, onto nitrocellulose membranes. After hybridization, biotin-labelled DNA bound to BYDV RNA on the nitrocellulose was detected with an avidin-alkaline phosphatase conjugate. BYDV RNA was readily detected with a sensitivity similar to that found with the same probe labelled with 32P by nick translation. Healthy plant extracts gave colourless spots.